The invention is directed to the use of NAD analogs and NADP analogs as enzyme cofactors in the measurement of enzyme activities, metabolites and substrates using enzymatic procedures which require the use of NAD and/or NADP cofactors for their determination.
NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) are enzyme cofactors which are widely used in the measurement of enzyme activities and metabolites. The popularity of these cofactors for analytical measurements arises from the fact that in their oxidized forms, i.e. NAD and NADP they have virtually no absorbance at wavelengths longer than about 320 nm. When they are reduced, for example during an enzyme or substrate assay, NADH and NADPH have absorbances in the ultraviolet region of the spectrum with a maximum at 340 nm. At this wavelength both reduced cofactors have a molar absorbtivity of 6.22xc3x97103. This unique property of the oxidized forms having virtually no absorbance at 340 nm and the reduced forms having a well defined absorbance maxima at 340 nm is the basis for their analytical use, especially in diagnostic test procedures.
Despite their popularity, these two cofactors are not without their shortcomings. One limitation is their somewhat weak oxidation potential, Eo=xe2x88x920.32 for NAD and NADP (Lehniger, A. L., Biochemistry, Worth Publishers, Inc., New York, 1970). For many analytes and substrates this is not a serious problem, but for some substrates the reaction has to be xe2x80x9cengineeredxe2x80x9d in order for it to proceed in the forward direction. An example of an xe2x80x9cengineeredxe2x80x9d reaction is the enzymatic measurement of ethanol using alcohol dehydrogenase according to the following reaction.
ethanol+NAD alcohol dehydrogenasexe2x86x92acetaldehyde+NADH+H+
To quantitatively measure ethanol in a sample the reaction needs to be at an alkaline pH, an excess of NAD has to be present and a means of removing the acetaldehyde must be incorporated into the reaction system to drive the reaction to completion. Typically a reactive primary amine e.g. 1,3-diamino-2-hydroxypropane is present to form a Schiff base with the acetaldehyde thereby removing it from solution (Kaufman et al., U.S. Pat. No. 5,141,854). The alkaline pH removes protons generated during ethanol oxidation which also helps drive the reaction in the forward direction. The alkaline pH, however, also brings with it other undesirable effects. NAD and NADP start to become unstable above pH 7 and their instability increases with increasing pH. In order to have an all liquid, single component reagent ready to use, with reasonable shelf life e.g. 18 months at 2xc2x0 to 8xc2x0 C., for measuring ethanol in analytical samples, the NAD must be stored in a separate container at a lower pH to maintain its stability. Thus at a minimum, a two vial reagent configuration is needed for an ethanol reagent to have reasonable shelf life stability. The NAD instability has been somewhat overcome by the addition of aliphatic zwitterionic secondary and tertiary amines, and maintaining the pH below 8 (Dorn et al., U.S. Pat. No. 5,804,403). But this technique still employs a two vial system which is more costly than single vial systems from both a labor and materials perspective. A trapping agent such as TRIS is also required to drive the reaction which further adds to costs. Other examples of metabolites which exhibit an unfavorable equilibrium for oxidation by NAD or NADP are glycerol, acetaldehyde, lactic acid and 3-hydroxybutyric acid.
A second limitation of NAD and NADP is their somewhat limited sensitivity. At 340 nm the molar absorbtivity of the reduced cofactors is 6.22xc3x97103. For most enzymes and metabolites measured by diagnostic procedures this is usually not a problem. With some diagnostic procedures, however, if a more sensitive cofactor were available better assay precision could be obtained or sample volumes could be reduced resulting in less interferences from endogenous serum sample components e.g. bilirubin, lipemia and hemoglobin. Examples of such clinical assays are the measurement of CK-MB activity which is an early and specific marker for patients having a myocardial infarction, some EST(trademark) assays where the analyte is very low e.g. digoxin and tetrahydrocannabinol (THC) and the determination of serum steroids and bile acids which are present in serum at very low concentrations (EMIT is a registered trademark of Dade Behring Inc., Deerfield, Ill.).
A third limitation of NAD and NADP is the somewhat limited wavelengths at which the reduced cofactors absorb. In the near-UV region of the spectrum the reduced cofactors have their maximum absorbance at 340 nm. At longer wavelengths their absorbances fall off rapidly and at wavelengths longer than 400 nm they exhibit no absorbance at all. To develop assays based on the reduced cofactors where a colored product absorbs in the visible region of the spectrum is desired, formazon dyes and diaphorases have been used. Diaphorases catalyze the reduction of an oxidized formazon dye using a reduced cofactor according to the following reaction.
xe2x80x83formazon dye+NAD(P)H diaphorasexe2x86x92reduced formazon dye+NAD(P) colorless dye highly colored dye
Assays for measuring serum triglycerides have been developed using formazon dyes to avoid the xe2x80x9cclearing effectxe2x80x9d, which is observed at 340 nm, and is due to the decrease in turbidity in the reaction mixture as the triglycerides in the sample are hydrolyzed by lipase(s) to fatty acids and glycerol.
Thus, given the inherent limitations for NAD and NADP mentioned above, there exists a need for enzyme cofactors with more favorable oxidation potentials, broader absorbance spectra in the near-UV and visible regions of the spectrum, and with higher sensitivities than NADH and NADPH.
NAD analogs and NADP analogs overcome many of the limitations mentioned above regarding NAD and NADP. For example, depending on the analog, many have more favorable Eo values which would facilitate oxidation reactions at lower pH (Anderson et al., J. Biol. Chem. 221, 1219, 1959). Kaplan et al. (J. Biol. Chem., 221, 823, 1956) found that the equilibrium constant for oxidation of ethanol to acetaldehyde using 3-acetylpyridine-NAD was 200 hundred times more favorable than with NAD. The absorbance maxima, depending on the reduced analog, absorbs not only in the ultraviolet region of the spectrum but well into the visible region as well (Siegel et al., Arch. Biochem. Biophys. 82, 288, 1959, and Stein et al., Biochemistry 2, 5, 1963).
In addition to the reduced analogs having ultraviolet and visible absorbance properties they also, depending on the analog, have significantly higher molar absorbtivities (see Siegel et al. and Stein et al. above). Siegel et al. observed that the molar absorbtivities of 3-acetylpyridine-NADH at 363 nm was 9.1xc3x97103 and 3-pyridinealdehyde-NADH at 358 nm was 9.3xc3x97103. Stein et al. found the molar absorbtivity of reduced thionicotinamide adenine dinucleotide at 398 mn was 11.9xc3x97103 and the corresponding thio-NADPH analog at 399 nm was 11.7xc3x97103. For a more complete listing of molar absorbtivities of NADH and NADPH analogs see Pyridine Nucleotide Coenzymes, edited by David Poulson and Olga Arramovic, Coenzymes and Cofactors, Vol III, Part A, John Wiley, New York, 1987.
In some developed procedures using NAD and NADP, Ueda et al. (U.S. Pat. No. 5,780,256) used a two coenzyme cycling technique, where the reagent contained two coenzymes e.g. NAD(P) and thio-NAD(P), to measure ammonia, bile acids (U.S. Pat. No. 5,286,627) and 3-hydroxybutyric in biological samples. In these methods one coenzyme is continuously recycled between two dehydrogenase enzymes while the other coenzyme is continually reduced to increase assay sensitivity. In another procedure, Makler (U.S. Pat. No. 5,124,141) found 3-acetylpyridine-NAD to be useful for diagnosing the presence lactic acid dehydrogenase originating from Plasmodium falcipanum (malaria) in human serum since human lactic acid dehydrogenases are unable to reduce 3-acetylpyridine-NAD.
Further discussions of NAD and NADP and their analogs can be found in several reviews e.g. The Pyridine Nucleotide Coenzymes, edited by Everse et al., Academic Press, New York, 1982, and Pyridine Nucleotide Coenzymes referred to above.
Separately, commonly assigned U.S. Pat. No. 5,801,006 discloses diagnostic kits and methods of measuring various metabolites and enzyme activities using the reduced forms of NADH analogs and NADPH analogs. In the ""006 patent, the reduced forms of the analogs demonstrate superior stability at pH""s of from about neutral to about pH 9 and afford the artisan with the ability to replace many two vial assay systems with one vial systems. The ""006 patent, however, is silent with regard to using the oxidized forms of the analogs in diagnostic tests. Furthermore, it could not be predicted from the ""006 patent that the mere substitution of NAD analogs and NADP analogs for NAD and NADP would yield unexpected benefits such as being able to provide single vial assays for measuring compounds such as ethanol, lactic acid and 3-hydroxybutyric acid at pH""s of from about 6 to 8.
In accordance with one aspect of the invention, the present invention includes a diagnostic reagent kit which includes a component of the formula: (I) 
wherein:
R1 is 
R2 is an aryl or heteroaryl;
Q is C or S;
T is O or S;
X is H. OR3 or H2PO4, where R3 is H, C1-4 alkyl, C1-4 haloalkyl, C1-4 substituted alkyl or halogen;
Y is O, S or NOH; and
Z is H, C1-6 alkyl, C1-6 haloalkyl, C1-6 substituted alkyl, NHL where L is H, OH, NH2 aryl or aralkyl; except that L is not H when R2 is adenine and Q is not S when Y is S.
In another preferred aspect of the invention, there is provided a method of quantifying the presence of an enzyme or analyte in a sample. The method includes:
a) contacting a sample with a compound of Formula I set forth above; and
b) measuring the change in absorbance or fluorescence resulting from said contacting step a).
Some preferred NAD analogs and NADP analogs which have been found to be useful for measuring enzyme activities and analytes using enzymes requiring these cofactors are 3-acetylpyridine adenine dinucleotide or 3-acetylpyridine-NAD; 3-acetylpyridine adenine dinucleotide phosphate or 3-acetylpyridine-NADP; 3-pyridinealdehyde adenine dinucleotide or 3-pyridine-aldehyde-NAD; 3-pyridinealdehyde adenine dinucleotide phosphate or 3-pyridinealdehyde-NADP; thionicotinamide adenine dinucleotide or thionicotinamide-NAD; and thionicotinamide adenine dinucleotide phosphate or thionicotinamide-NADP.
In preferred aspects of the invention, the NAD analogs and NADP analogs are included in kits and methods for determining the presence of metabolite or enzyme activities in an analytical sample. For example, glucose, triglycerides, bile acids, lactic acid, 3-hydroxybutyric acid, glycerol, xcex1-glycerophosphate, ethanol, creatine kinase activity, lactate dehydrogenase activity and glucose-6-phosphate dehydrogenase activity (EMIT(trademark) assays) can be determined in analytical samples using the analogs. One of the advantages of the present invention is that certain NAD analogs and NADP analogs such as 3-acetylpyridine-NAD, 3-acetylpyridine-NADP, 3-pyridinealdehyde-NAD, 3-pyridinealdehyde-NADP, thionicotinamide-NAD and thionicotinamide-NADP have more favorable oxidation potentials than NAD and NADP thus allowing oxidation of analytes by the analogs to proceed under conditions which is not feasible with NAD and NADP. For example oxidation reactions can often be carried out at a lower pH and often eliminating the need to remove one of the forward reaction products. Examples of such analytes are ethanol, lactic acid and 3-hydroxybutyric acid. The lower pH allows kits to be configured as ready-to-use single vial liquid reagents which simplifies the manufacturing process and makes reagents more convenient to use by the end user.
Another advantage of the analogs is that in their reduced form they have absorbances in the near-UV and also in the visible region of the spectrum. For example the reduced analogs 3-acetylpyridine-NADH, 3-acetylpyridine-NADPH, 3-pyridinealdehyde-NADH, and 3-pyridinealdehyde-NADPH have appreciable absorbances at 405 nm with molar absorbtivities of approximately 2xc3x97103. Thionicotinamide-NADH and thionicotinamide-NADPH have absorbance maxima at 398 nm with molar absorbtivities of about 11.9xc3x97103.
A third advantage of certain NAD analogs and NADP analogs is the increased sensitivity of the reduced forms compared with NADH and NADPH. For example 3-acetylpyridine-NADH, 3-acetylpyridine-NADPH have absorbance maxima at 363 nm with molar absorbtivities of 9.4xc3x97103 which at this wavelength is 1.5 times more sensitive than NADH and NADPH at their absorbance maxima at 340 nm which is 6.22xc3x97103. Similarly 3-pyridinealdehyde-NADH has absorbance maxima at 358 nm with a molar absorbtivity of 9.9xc3x97103. As mentioned above the thionicotinamide analogs have sensitivities at 398 nm nearly twice NADH and NADPH at 340 nm. For enzyme and analyte assays requiring increased sensitivity, the analogs provide approximately 1.5 to 2 fold increased sensitivity and at longer wavelengths which also reduces spectral interference form e.g. endogenous sample interferrents such as lipemia (triglycerides), bilirubin and hemoglobin found in serum.
One of the advantages of the methods of the present invention is that they allow the artisan to measure various metabolites at pH ranges around slightly acidic, neutral to slightly alkaline e.g. up to pH 8.0 or greater when compared to NAD or NADP. Thus, economical single vial systems can be prepared.
In one preferred aspect the invention is directed to diagnostic kits containing NAD analogs and NADP analogs. The analogs are of the Formula: (I) 
wherein:
R1 is 
R2 is an aryl or heteroaryl;
Q is C or S;
T is O or S;
X is H, OR3 or H2PO4, where R3 is H, C1-4 alkyl, C1-4 haloalkyl, C1-4 substituted alkyl or halogen;
Y is O, S or NOH; and
Z is H, C1-6 alkyl, C1-6 haloalkyl, C1-6 substituted allyl, NHL where L is H, OH, NH2 aryl or aralkyl; except that L is not H when R2 is adenine and Q is not S when Y is S.
Within Formula (I), R1 is preferably selected from among the group: 
Alternatively, R1 can be selected from among: 
Preferably, R2 is a an adenine such as 
Alternatively, R2 can be a substituted adenine, a substituted or unsubstituted member of the group consisting of xanthines, thioxanthines, hypoxanthines, guanines or other fused heterocyclic ring structures, aryls, substituted aryls, etc.
Also within Formula (I), X is preferably OH or H2PO4 and each T is O. Preferred compounds for inclusion with the kits and methods describe herein in accordance with Formula (I) include 3-acetylpyridine-NAD, 3-acetylpyridine-NADP, 3-pyridinealdehyde-NAD, 3-pyridinealdehyde-NADP, thionicotinamide-NAD and thionicotinamide-NADP. These compounds can be synthesized using standard organic chemistry techniques, or, if desired, purchased from commercial suppliers such as Sigma Chemical Co. It is contemplated that the kits of the present invention will include the compounds of Formula (I) in amounts ranging from about 0.01 to about 10 mmol/L.
The kits and methods of the present invention are useful in the measurement of various enzyme activities and analytes. A non-limiting list of such materials, such as substrates or metabolites and enzyme activities, which can be measured using the analogs described herein include creatine kinase, glucoses-6-phosphate dehydrogenase (EMIT(trademark) assays), lactate dehydrogenase, ethanol, glucose, glycerol, xcex1-glycerophosphate, triglycerides, bile acids, lactic acid and 3-hydroxybutyric acid.
In additional aspects of the invention, the diagnostic kits also may include an enzyme such as NAD and/or NADP-dependent enzymes. A non-limiting list of such enzymes includes lactate dehydrogenase, alcohol dehydrogenase, 3-hydroxybutyric acid dehydrogenase, glucose dehydrogenase, and glucose-6-phosphate dehydrogenase, xcex1-glycerophosphate dehydrogenase and 3-xcex1-hydroxysteroid dehydrogenase. Other enzymes will be apparent to those of ordinary skill. The enzymes will be present in sufficient amounts to provide enzyme activities from about 0.05 to about 150 Units/ml of reagent containing solution. It will be understood that the actual amounts of enzyme activity will depend upon the enzyme(s) included in the kit and target metabolite or enzyme activity sought to be measured.
Since the NAD/NADP analogs of Formula (I) are not the xe2x80x9cnaturalxe2x80x9d cofactors commonly found in biological systems, the functionality of the analogs with dehydrogenase enzymes must be determined.
The kits of the present invention can be prepared in either wet or dry form, including lyophilized form, depending upon the needs of the user. The kits can also include a suitable buffer such as tris(hydroxymethyl)aminomethane, [3-(N-morpholino)-2-hydroxypropane-sulfonic acid] and trapping compounds such as primary and secondary amines, and diamines which may interact with aldehyde and ketone groups formed during oxidation of hydroxyl groups. The purpose of having trapping compounds is to drive the reaction towards completion by removing one of the end products from the reaction. It will be apparent to those of ordinary skill in the art that the buffer included will depend upon the preference of the artisan and will also be selected based on the metabolite or enzyme activity sought to be measured. It is contemplated, however, that there will be sufficient buffer in the kits, i.e. from about 0.01 to about 1.0 mol/liter, and the analog containing solution will have a pH of from about 5 to 10. Also the trapping compounds which may be used for metabolites such as lactic acid, 3-hydroxybutyric acid, glycerol, xcex1-glycerophosphate and ethanol will be used at concentrations sufficient to drive the reaction far enough in the forward direction to obtain the desired assay dynamic range. Concentrations will range from about 0.01 mol/liter to about 1.0 mol/liter. A competitive enzyme inhibitor such as pyrazole may be added to the ethanol reagent to extend the dynamic range of the assay.
In still further aspects of the invention, the diagnostic kits can contain a substrate such as creatine phosphate, adenosine-5xe2x80x2-diphosphate, glucose, glucose-6-phosphate, lactic acid and adenosine triphosphate. The substrates will be present in amounts from about 0.1 mmol/liter to about 1000 mmol/liter. If desired, the kits can be prepared to include a suitable antimicrobial such as sodium azide, Kathon, Bronopol or parabens. Such antimicrobials can be present in amounts ranging from about 0.01 to about 0.5% by wt.
Reagents in accordance with the present invention can be configured in several different formats. A single vial may be prepared which contains all necessary components including an antimicrobial, buffer, and components to stabilize the coupling enzyme(s), if present. For convenience, a single vial ready-to-use liquid reagent is preferred with a storage temperature of about +2xc2x0 to +8xc2x0 C. (refrigerator storage). Alternatively, the reagents may be prepared as a two component system or even a three or more component system and as powder (dry-fill) or lyophilizate. Having components of the reagent in separate vials or bottles usually results in better component stability, but may be deemed less convenient by some end users.